Splice variant of head trauma induced cytoplasmatic calcium binding protein

ABSTRACT

ANIC-BP-1B polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing ANIC-BP-1B polypeptides and polynucleotides in diagnostic assays.

FIELD OF THE INVENTION

This invention relates to nucleic acid and amino acid sequences of a human Acute Neuronal Induced Calcium-Binding Protein (ANIC-BP)-like protein splice variant and to the use of these sequences, sometimes hereinafter referred to as “ANIC-BP-1B”. The invention as well provides expression vectors, host cells and antibodies. In addition the invention provides methods for producing the protein and for treating or preventing disorders associated with the expression of the protein. The invention also relates to inhibiting or activating the action of such polynucleotides and polypeptides.

BACKGROUND OF THE INVENTION

Stroke and acute head trauma, multiple sclerosis and spinal cord injury are diseases for which so far there is no therapy available. Stroke is the third leading cause of death and the burden for the patients as well as for the social systems are enormous. In case of ischaemic stroke, which accounts for most of the strokes, blood vessel blockade in the brain is an initial event. Head trauma accidents are the leading disease of young people in the western world. A much smaller number of patients is affected by haemorhagic stroke caused by mechanical impact and artery rupture. It is a common feature, that approved pharmaceuticals are hardly available. Currently available treatment approaches are based upon pathophysiologic concepts derived from experimental work with focal cerebral ischaemia. These include pharmacological strategies for arterial recanalisation, inhibition of inflammatory processes and neural protection. Further work with arterial reperfusion strategies is under way. Early clinical studies with polymorphonuclear leukocyte-dependent endothelial adhesion receptor antagonists are being completed, but a strategy has yet to emerge. However any strategy addressing only a single step in the ischaemic cascade is likely to produce a modest benefit only. Therefor future therapies most properly will be based on combination therapies. A combination of low-dose acetylsalicylic acid (ASA) and modified-release dipyridamole has been shown to be additive in the secondary prevention of stroke (Tijssen et al., Int.J.Clin. Pract., suppl. 91, 14–16, 1997). Another combination which is currently proposed for clinical evaluation is tPA plus an effective neuroprotective agent. However the results of these studies are fare from being highly effective. Hence, there is urgent need to learn more about the pathophysiological mechanisms in order to provide drug targets that could be used to develop new drugs and establish more generally useful regimens for the treatment of patients suffering from acute neurodamaging effects and disorders such as multiple sclerosis.

This invention does focus on Ca²⁺-binding proteins since there has been considerable evidence for a role of Ca²⁺-binding proteins in neuroprotection. Although the precise function of calcium binding proteins (CaBPs) is not definitively known, it has been proposed that CaBPs act to buffer intracellular Ca²⁺ levels. Since Ca²⁺ overload activates biochemical processes leading to proteolysis and mitochondrial malfunction, this buffering capacity of CaBPs may have a protective effect against excitotoxic neuronal injury (Heizmann et al., TINS, 15, 259–64, 1992). A variety of evidences exists to support this proposed role of CaBPs in modulation of Ca²⁺ levels and in neuroprotection.

The major Ca²⁺-binding proteins (CaBPs) expressed in the central nervous system (parvalbumin, calbindin-D28K, and calretinin) have a very unusual and selective pattern of expression in various neuronal populations. Among neurons that do express CaBPs, most express only one type, although a small number of neurons express more than one of the major calcium binding proteins. There is growing evidence that the presence or absence of CaBPs in particular cell types underlies the phenomenon known as selective vulnerability. Selective vulnerability is a property of specific types of neurons to die in response to particular types of central nervous system (CNS) injury. For example, CA1 hippocampal neurons are selectively vulnerable to global ischemia, cerebellar Purkinje cells are selectively vulnerable to head trauma, stroke, and fetal alcohol exposure, and neurons in the substantia nigra are selectively vulnerable in Parkinson's Disease. An effort has been made to link selective neuronal vulnerability to the expression patterns of various CaBPs and some authors report that high levels of CaBPs are found in neuronal populations that are selectively vulnerable to injury, while others report high CaBP levels in neuronal populations that are selectively resistant to injury. For example, neurons expressing high levels of parvalbumin are reported to be selectively vulnerable to AMPA-induced toxicity (Weiss et al., Neurol., 40, 1288–1292, 1990), whereas cultured hippocampal neurons expressing high levels of calbindin-D28K are reported to be selectively resistant to glutamate-induced toxicity (Baimbridge et al., TINS, 15, 303–8, 1992). Similarly, hippocampal neurons expressing high levels of calretinin are resistant to toxic doses of the excitotoxins glutamate, NMDA, kainate, and quisqualate (Winsky et al., in: Novel Calcium-Binding Proteins, 277–300, 1991).

CaBPs have also been shown to have altered expression in various CNS disease states, but again the results are inconsistent about whether CaBP expression is related to selective vulnerability or selective resistance to injury. Neurons expressing calbindin-D28K are reported to be selectively vulnerable in Alzheimer's Disease (lacopino et al. PNAS, 87, 4078–82, 1990, Hof et al. Exp. Neurol., 111, 293–301, 1991) and Huntington's Disease (Kiyama et al., Brain Res., 526, 303–07, 1990), although calbindin-D28K-expressing neurons in the substantia nigra are not selectively vulnerable in Parkinson's Disease (Yamada et al., Brain Res., 526, 303–07, 1990). In a gerbil model of global ischemia, the presence of parvalbumin in certain hippocampal cell types has been shown to be positively associated with survival (Tortosa et al., Neurosci., 1, 33–43, 1993), although another study suggested that parvalbumin-expressing hippocampal interneurons are selectively vulnerable in Alzheimer's Disease (Brady et al., Neurosci., 80, 1113–25, 1997).

Mice with a knockout of the calbindin gene show functional deficits (e.g. ataxia) that suggest severe dysfunction in neurons normally expressing this CaBP (e.g., cerebellar Purkinje cells) despite the fact that these neurons appear morphologically normal. This finding suggests that CaBPs are vital to cellular activity patterns (Airaksinen et al., PNAS, 94, 1488–93, 1997). In addition, retroviral infection of motoneurons with calbindin-D28k has been shown to have neuroprotective effects against toxicity induced by IgG from patients with amyotrophic lateral sclerosis (Ho et al., PNAS, 93, 6796–801, 1996) and transfection with calbindin-D28k has been shown to protect PC12 cells from toxicity due to serum withdrawl, glutamate exposure, and the neurotoxin MPP+ (McMahon et al., Molec. Brain Res., 526, 303–07, 1998).

In conclusion, there is considerable information regarding a role for calcium binding proteins in neurodegeneration. It is certain that some CaBPs provide protection and others cause selective vulnerability, however it is not yet clear whether the expression of certain CaBPs within different neuronal populations results in different functional responses of a given CaBP. Another observation is that the severity of different types of CNS injury may affect the apparent neuroprotective efficacy of CaBPs—i.e., one CaBP may confer resistance in an injury model involving a mild injury, but may be unable to buffer Ca²⁺ increases in more severe CNS injuries. Thus there is considerable evidence that CaBPs confer resistance as well as vulnerability in CNS injury process, but the mechanism of this involvement and the regulation of the response has still to be worked out.

A gene familly comprising the functionally unidentified gene (MO25) was recently isolated from a mouse derived cDNA library (Miyamoto et al., Mol. Reprod. Dev., 34, 1–7, 1993). The library was constructed from RNA isolated from an early embrionic mouse. The predicted amino acid sequence for Mo25 revealed that the MO25 gene may have structural homology with Ca2+ binding proteins and lack membrane spanning domaines, indicating that the protein might be involved in cytosolic development of the unfertilized egg. However the real function of this protein remains unknown. Another Mo25 like gene, has been cloned from a Drosophila cDNA library (Nozaki et al. DNA Cell Biol., 15, 505–09, 1996). The deduced amino acid sequence of the Mo25 cDNA had 69.3% identity with mouse Mo25 homologe. A homologue in Saccharomyces cerevisiae encoded in an open reading frame near the calcineurin B subunit gene. Most recently another gene has been isolated from Aspergillus hym A mutants (Karos et al., Mol. Gen Genet., 260, 510–521, 1999) and turned out to correspond to the homologues in yeast, plants, fly, worm, fish, mice and man. A cellular function for the Hym protein has not yet been defined in any of the decribed organisms. As with many other other proteins where the functional contribution is only partially understood the drug discovery process is currently undergoing a fundamental revolution as it embraces “functional genomics”, that is, high throughput genome- or gene-based biology. This approach as a means to identify genes and gene products as therapeutic targets is rapidly superceding earlier approaches based on “positional cloning”. A phenotype, that is a biological function or genetic disease, would be identified and this would then be tracked back to the responsible gene, based on its genetic map position.

Functional genomics relies heavily on high-throughput DNA sequencing technologies and the various tools of bioinformatics to identify gene sequences of potential interest from the many molecular biology databases now available. There is a continuing need to identify and characterise further genes and their related polypeptides/proteins, as targets for drug discovery. The discovery of a new human ANIC-BP-like protein splice variant and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in the diagnosis, treatment, and prevention of reproductive, immunological, vesicle trafficking, nervous system, developmental, and neoplastic disorders.

SUMMARY OF THE INVENTION

The present invention relates to the discovery of a new human ANIC-BP —like protein splice variant in particular ANIC-BP-1B polypeptides and ANIC-BP-1B polynucleotides, recombinant materials and methods for their production. Such polypeptides and polynucleotides are of interest in relation to methods of treatment of certain diseases, including, but not limited to stroke and acute head trauma, Parkinson's disease, Alzheimer disease, multiple sclerosis and spinal cord injury, hereinafter referred to as “diseases of the invention”. In a further aspect, the invention relates to methods for identifying agonists and antagonists (e.g., inhibitors) using the materials provided by the invention, and treating conditions associated with ANIC-BP-1B imbalance with the identified compounds. In a still further aspect, the invention relates to diagnostic assays for detecting diseases associated with inappropriate ANIC-BP-1B activity or levels.

DESCRIPTION OF THE INVENTION

In a first aspect, the present invention relates to ANIC-BP-1B polypeptides. Such polypeptides include:

-   -   (a) an isolated polypeptide encoded by a polynucleotide         comprising the sequence of SEQ ID NO:1;     -   (b) an isolated polypeptide comprising a polypeptide sequence         having at least 95%, 96%, 97%, 98%, or 99% identity to the         polypeptide sequence of SEQ ID NO:2;     -   (c) an isolated polypeptide comprising the polypeptide sequence         of SEQ ID NO:2;     -   (d) an isolated polypeptide having at least 95%, 96%, 97%, 98%,         or 99% identity to the polypeptide sequence of SEQ ID NO:2;     -   (e) the polypeptide sequence of SEQ ID NO:2; and     -   (f) an isolated polypeptide having or comprising a polypeptide         sequence that has an Identity Index of 0.95, 0.96, 0.97, 0.98,         or 0.99 compared to the polypeptide sequence of SEQ ID NO:2;     -   (g) fragments and variants of such polypeptides in (a) to (f).

Polypeptides of the present invention are believed to be members of the ANIC-BP family of polypeptides. They are of interest because a recently described gene member of this family, called ANIC-BP was found upregulated in a rat model of head trauma as discovered with the technique of mRNA diffferential display. The variant protein could serve as an novel drug target.

The biological properties of the ANIC-B-1B are hereinafter referred to as “biological activity of ANIC-BP-1B “or” ANIC-BP-1B activity”. Preferably, a polypeptide of the present invention exhibits at least one biological activity of ANIC-BP-1B.

Polypeptides of the present invention also includes variants of the aforementioned polypeptides, including all allelic forms and splice variants. Such polypeptides vary from the reference polypeptide by insertions, deletions, and substitutions that may be conservative or non-conservative, or any combination thereof. Particularly preferred variants are those in which several, for instance from 50 to 30, from 30 to 20, from 20 to 10, from 10 to 5, from 5 to 3, from 3 to 2, from 2 to 1 or 1 amino acids are inserted, substituted, or deleted, in any combination.

Preferred fragments of polypeptides of the present invention include an isolated polypeptide comprising an amino acid sequence having at least 30, 50 or 100 contiguous amino acids from the amino acid sequence of SEQ ID NO: 2, or an isolated polypeptide comprising an amino acid sequence having at least 30, 50 or 100 contiguous amino acids truncated or deleted from the amino acid sequence of SEQ ID NO: 2. Preferred fragments are biologically active fragments that mediate the biological activity of ANIC-BP-1B, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also preferred are those fragments that are antgenic or immunogenic in an animal, especially in a human.

Fragments of the polypeptides of the invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, these variants may be employed as intermediates for producing the full-length polypeptides of the invention. The polypeptides of the present invention may be in the form of the “mature” protein or may be a part of a larger protein such as a precursor or a fusion protein. It is often advantageous to include an additional amino acid sequence that contains secretory or leader sequences, pro-sequences, sequences that aid in purification, for instance multiple histidine residues, or an additional sequence for stability during recombinant production.

Polypeptides of the present invention can be prepared in any suitable manner, for instance by isolation form naturally occurring sources, from genetically engineered host cells comprising expression systems (vide infra) or by chemical synthesis, using for instance automated peptide synthesisers, or a combination of such methods. Means for preparing such polypeptides are well understood in the art.

In a further aspect, the present invention relates to ANIC-BP-1B polynucleotides. Such polynucleotides include:

-   -   (a) an isolated polynucleotide comprising a polynucleotide         sequence having at least 95%, 96%, 97%, 98%, or 99% identity to         the polynucleotide squence of SEQ ID NO:1;     -   (b) an isolated polynucleotide comprising the polynucleotide of         SEQ ID NO:1;     -   (c) an isolated polynucleotide having at least 95%, 96%, 97%,         98%, or 99% identity to the polynucleotide of SEQ ID NO:1;     -   (d) the isolated polynucleotide of SEQ ID NO:1;     -   (e) an isolated polynucleotide comprising a polynucleotide         sequence encoding a polypeptide sequence having at least 95%,         96%, 97%, 98%, or 99% identity to the polypeptide sequence of         SEQ ID NO:2;     -   (f) an isolated polynucleotide comprising a polynucleotide         sequence encoding the polypeptide of SEQ ID NO:2;     -   (g) an isolated polynucleotide having a polynucleotide sequence         encoding a polypeptide sequence having at least 95%, 96%, 97%,         98%, or 99% identity to the polypeptide sequence of SEQ ID NO:2;     -   (h) an isolated polynucleotide encoding the polypeptide of SEQ         ID NO:2;     -   (i) an isolated polynucleotide having or comprising a         polynucleotide equence that has an Identity Index of 0.95, 0.96,         0.97, 0.98, or 0.99 compared to the polynucleotide sequence of         SEQ ID NO:1;     -   (j) an isolated polynucleotide having or comprising a         polynucleotide sequence encoding a polypeptide sequence that has         an Identity Index of 0.95, 0.96, 0.97, 0.98, or 0.99 compared to         the polypeptide sequence of SEQ ID NO:2; and polynucleotides         that are fragments and variants of the above mentioned         polynucleotides or that are complementary to above mentioned         polynucleotides, over the entire length thereof.

Preferred fragments of polynucleotides of the present invention include an isolated polynucleotide comprising an nucleotide sequence having at least 15, 30, 50 or 100 contiguous nucleotides from the sequence of SEQ ID NO: 1, or an isolated polynucleotide comprising an sequence having at least 30, 50 or 100 contiguous nucleotides truncated or deleted from the sequence of SEQ ID NO: 1.

Preferred variants of polynucleotides of the present invention include splice variants, allelic variants, and polymorphisms, including polynucleotides having one or more single nucleotide polymorphisms (SNPs).

Polynucleotides of the present invention also include polynucleotides encoding polypeptide variants that comprise the amino acid sequence of SEQ ID NO:2 and in which several, for instance from 50 to 30, from 30 to 20, from 20 to 10, from 10 to 5, from 5 to 3, from 3 to 2, from 2 to 1 or 1 amino acid residues are substituted, deleted or added, in any combination.

In a further aspect, the present invention provides polynucleotides that are RNA transcripts of the DNA sequences of the present invention. Accordingly, there is provided an RNA polynucleotide that:

-   -   a) comprises an RNA transcript of the DNA sequence encoding the         polypeptide of SEQ ID NO:2;     -   b) is the RNA transcript of the DNA sequence encoding the         polypeptide of SEQ ID NO:2;     -   c) comprises an RNA transcript of the DNA sequence of SEQ ID         NO:1; or     -   d) is the RNA transcript of the DNA sequence of SEQ ID NO:1; and         RNA polynucleotides that are complementary thereto.

The polynucleotide sequence of SEQ ID NO:1 shows homology with Hym A (Nozaki et al. DNA cell Biol., 15, 505–09, 1996), Mo25 (Karos et al., Mol. Gen Genet., 260, 510–521, 1999) and ANIC-BP. A comparison of the amino acid sequences of ANIC-BP and ANIC-BP-1B protein (SEQ ID NO:2) has been performed and indicates considerable differences in the C-terminal portion (amino acid portion 315 to 338 of SEQ ID NO:2) of said molecules.

The polynucleotide sequence of SEQ ID NO:1 is a cDNA sequence that encodes the polypeptide of SEQ ID NO:2. The polynucleotide sequence encoding the polypeptide of SEQ ID NO:2 may be identical to the polypeptide encoding sequence of SEQ ID NO:1 or it may be a sequence other than SEQ ID NO:1, which, as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO:2. The polypeptide of the SEQ ID NO:2 is related to other proteins of the *Calcium binding protein family, having homology and/or structural similarity with ANIC-BP.

Preferred polypeptides and polynucleotides of the present invention are expected to have, inter alia, similar biological functions/properties to their homologous polypeptides and polynucleotides. Furthermore, preferred polypeptides and polynucleotides of the present invention have at least an 5 ANIC-BP-1B activity.

Polynucleotides of the present invention may be obtained using standard cloning and screening techniques from a cDNA library derived from mRNA of human monocytes activated cells, (see for instance, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). Polynucleotides of the invention can also be obtained from natural sources such as genomic DNA libraries or can be synthesized using well known and commercially available techniques.

When polynucleotides of the present invention are used for the recombinant production of polypeptides of the present invention, the polynucleotide may include the coding sequence for the mature polypeptide, by itself, or the coding sequence for the mature polypeptide in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro-protein sequence, or other fusion peptide portions. For example, a marker sequence that facilitates purification of the fused polypeptide can be encoded. In certain preferred embodiments of this aspect of the invention, the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al, Proc Natl Acad Sci USA (1989) 86:821–824, or is an HA tag. The polynucleotide may also contain non-coding 5′ and 3′ sequences, such as transcribed, non-translated sequences, splicing and polyadenylation signals, ribosome binding sites and sequences that stabilize mRNA.

Polynucleotides that are identical, or have sufficient identity to a polynucleotide sequence of SEQ ID NO:1, may be used as hybridization probes for cDNA and genomic DNA or as primers for a nucleic acid amplification reaction (for instance, PCR). Such probes and primers may be used to isolate full-length cDNAs and genomic clones encoding polypeptides of the present invention and to isolate cDNA and genomic clones of other genes (including genes encoding paralogs from human sources and orthologs and paralogs from species other than humans) that have a high sequence similarity to SEQ ID NO:1, typically at least 95% identity. Preferred probes and primers will generally comprise at least 15 nucleotides, preferably, at least 30 nucleotides and may have at least 50, if not at least 100 nucleotides. Particularly preferred probes will have between 30 and 50 nucleotides. Particularly preferred primers will have between 20 and 25 nucleotides.

A polynucleotide encoding a polypeptide of the present invention, including homologs from species other than humans, may be obtained by a process comprising the steps of screening a library under stringent hybridization conditions with a labeled probe having the sequence of SEQ ID NO: 1 or a fragment thereof, preferably of at least 15 nucleotides; and isolating full-length cDNA and genomic clones containing said polynucleotide sequence. Such hybridization techniques are well known to the skilled artisan. Preferred stringent hybridization conditions include overnight incubation at 420° C. in a solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA; followed by washing the filters in 0.1×SSC at about 650° C. Thus the present invention also includes isolated polynucleotides, preferably with a nucleotide sequence of at least 100, obtained by screening a library under stringent hybridization conditions with a labeled probe having the sequence of SEQ ID NO:1 or a fragment thereof, preferably of at least 15 nucleotides.

The skilled artisan will appreciate that, in many cases, an isolated cDNA sequence will be incomplete, in that the region coding for the polypeptide does not extend all the way through to the 5′ terminus. This is a consequence of reverse transcriptase, an enzyme with inherently low “processivity” (a measure of the ability of the enzyme to remain attached to the template during the polymerisation reaction), failing to complete a DNA copy of the mRNA template during first strand cDNA synthesis.

There are several methods available and well known to those skilled in the art to obtain full-length cDNAs, or extend short cDNAs, for example those based on the method of Rapid Amplification of cDNA ends (RACE) (see, for example, Frohman et al., Proc Nat Acad Sci USA 85, 8998–9002, 1988). Recent modifications of the technique, exemplified by the Marathon (trade mark) technology (Clontech Laboratories Inc.) for example, have significantly simplified the search for longer cDNAs. In the Marathon (trade mark) technology, cDNAs have been prepared from mRNA extracted from a chosen tissue and an ‘adaptor’ sequence ligated onto each end. Nucleic acid amplification (PCR) is then carried out to amplify the “missing” 5′ end of the cDNA using a combination of gene specific and adaptor specific oligonucleotide primers. The PCR reaction is then repeated using ‘nested’ primers, that is, primers designed to anneal within the amplified product (typically an adaptor specific primer that anneals further 3′ in the adaptor sequence and a gene specific primer that anneals further 5′ in the known gene sequence). The products of this reaction can then be analysed by DNA sequencing and a full-length cDNA constructed either by joining the product directly to the existing cDNA to give a complete sequence, or carrying out a separate full-length PCR using the new sequence information for the design of the 5′ primer.

Recombinant polypeptides of the present invention may be prepared by processes well known in the art from genetically engineered host cells comprising expression systems. Accordingly, in a further aspect, the present invention relates to expression systems comprising a polynucleotide or polynucleotides of the present invention, to host cells which are genetically engineered with such expression sytems and to the production of polypeptides of the invention by recombinant techniques. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention.

For recombinant production, host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention. Polynucleotides may be introduced into host cells by methods described in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology (1986) and Sambrook et al. (ibid). Preferred methods of introducing polynucleotides into host cells include, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.

Representative examples of appropriate hosts include bacterial cells, such as Streptococci, Staphylococci, E. coli, Streptomyces and Bacillus subtilis cells; fungal cells, such as yeast cells and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells; and plant cells.

A great variety of expression systems can be used, for instance, chromosomal, episomal and virus-derived systems, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. The expression systems may contain control regions that regulate as well as engender expression. Generally, any system or vector that is able to maintain, propagate or express a polynucleotide to produce a polypeptide in a host may be used. The appropriate polynucleotide sequence may be inserted into an expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al., (ibid). Appropriate secretion signals may be incorporated into the desired polypeptide to allow secretion of the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space or the extracellular environment. These signals may be endogenous to the polypeptide or they may be heterologous signals.

If a polypeptide of the present invention is to be expressed for use in screening assays, it is generally preferred that the polypeptide be produced at the surface of the cell. In this event, the cells may be harvested prior to use in the screening assay. If the polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the polypeptide. If produced intracellularly, the cells must first be lysed before the polypeptide is recovered.

Polypeptides of the present invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during intracellular synthesis, isolation and/or purification.

Polynucleotides of the present invention may be used as diagnostic reagents, through detecting mutations in the associated gene. Detection of a mutated form of the gene characterised by the polynucleotide of SEQ ID NO:1 in the cDNA or genomic sequence and which is associated with a dysfunction will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, or susceptibility to a disease, which results from under-expression, over-expression or altered spatial or temporal expression of the gene. Individuals carrying mutations in the gene may be detected at the DNA level by a variety of techniques well known in the art.

Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy material. The genomic DNA may be used directly for detection or it may be amplified enzymatically by using PCR, preferably RT-PCR, or other amplification techniques prior to analysis. RNA or cDNA may also be used in similar fashion. Deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to labeled ANIC-BP-1B nucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures. DNA sequence difference may also be detected by alterations in the electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing (see, for instance, Myers et al., Science (1985) 230:1242). Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and S1 protection or the chemical cleavage method (see Cotton et al., Proc Natl Acad Sci USA (1985) 85: 4397–4401).

An array of oligonucleotides probes comprising ANIC-BP-1B polynucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e.g., genetic mutations. Such arrays are preferably high density arrays or grids. Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability, see, for example, M. Chee et al., Science, 274, 610–613 (1996) and other references cited therein.

Detection of abnormally decreased or increased levels of polypeptide or mRNA expression may also be used for diagnosing or determining susceptibility of a subject to a disease of the invention. Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, nucleic acid amplification, for instance PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods. Assay techniques that can be used to determine levels of a protein, such as a polypeptide of the present invention, in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays.

Thus in another aspect, the present invention relates to a diagnostic kit comprising:

-   -   (a) a polynucleotide of the present invention, preferably the         nucleotide sequence of SEQ ID NO: 1, or a fragment or an RNA         transcript thereof;     -   (b) a nucleotide sequence complementary to that of (a);     -   (c) a polypeptide of the present invention, preferably the         polypeptide of SEQ ID NO:2 or a fragment thereof; or     -   (d) an antibody to a polypeptide of the present invention,         preferably to the polypeptide of SEQ ID NO:2.

It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component. Such a kit will be of use in diagnosing a disease or susceptibility to a disease, particularly diseases of the invention, amongst others.

The polynucleotide sequences of the present invention are valuable for chromosome localization studies. The sequence is specifically targeted to, and can hybridize with, a particular location on an individual human chromosome. The mapping of relevant sequences to chromosomes according to the present invention is an important first step in correlating those sequences with gene associated disease. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found in, for example, V. McKusick, Mendelian Inheritance in Man (available on-line through Johns Hopkins University Welch Medical Library). The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (co-inheritance of physically adjacent genes). Precise human chromosomal localizations for a genomic sequence (gene fragment etc.) can be determined using Radiation Hybrid (RH) Mapping (Walter, M. Spillett, D., Thomas, P., Weissenbach, J., and Goodfellow, P., (1994) A method for constructing radiation hybrid maps of whole genomes, Nature Genetics 7, 22–28). A number of RH panels are available from Research Genetics (Huntsville, Ala., USA) e.g. the GeneBridge4 RH panel (Hum Mol Genet 1996 March; 5(3):339–46 A radiation hybrid map of the human genome. Gyapay G, Schmitt K, Fizames C, Jones H, Vega-Czarny N, Spillett D, Muselet D, Prud'Homme J F, Dib C, Auffray C, Morissette J, Weissenbach J, Goodfellow PN). To determine the chromosomal location of a gene using this panel, 93 PCRs are performed using primers designed from the gene of interest on RH DNAs. Each of these DNAs contains random human genomic fragments maintained in a hamster background (human/hamster hybrid cell lines). These PCRs result in 93 scores indicating the presence or absence of the PCR product of the gene of interest. These scores are compared with scores created using PCR products from genomic sequences of known location. This comparison is conducted on the world wide web at www.genome.wi.mit.edu.

The polynucleotide sequences of the present invention are also valuable tools for tissue expression studies. Such studies allow the determination of expression patterns of polynucleotides of the present invention which may give an indication as to the expression patterns of the encoded polypeptides in tissues, by detecting the mRNAs that encode them. The techniques used are well known in the art and include in situ hydridisation techniques to clones arrayed on a grid, such as cDNA microarray hybridisation (Schena et al, Science, 270, 467470, 1995 and Shalon et al, Genome Res, 6, 639–645, 1996) and nucleotide amplification techniques such as PCR. A preferred method uses the TAQMAN (Trade mark) technology available from Perkin Elmer. Results from these studies can provide an indication of the normal function of the polypeptide in the organism. In addition, comparative studies of the normal expression pattern of mRNAs with that of mRNAs encoded by an alternative form of the same gene (for example, one having an alteration in polypeptide coding potential or a regulatory mutation) can provide valuable insights into the role of the polypeptides of the present invention, or that of inappropriate expression thereof in disease. Such inappropriate expression may be of a temporal, spatial or simply quantitative nature. The polypeptides of the present invention are expressed in human monocyte actvated cells.

A further aspect of the present invention relates to antibodies. The polypeptides of the invention or their fragments, or cells expressing them, can be used as immunogens to produce antibodies that are immunospecific for polypeptides of the present invention. The term “immunospecific” means that the antibodies have substantially greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the prior art.

Antibodies generated against polypeptides of the present invention may be obtained by administering the polypeptides or epitope-bearing fragments, or cells to an animal, preferably a non-human animal, using routine protocols. For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler, G. and Milstein, C., Nature (1975) 256:495497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., Immunology Today (1983) 4:72) and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, 77–96, Alan R. Liss, Inc., 1985).

Techniques for the production of single chain antibodies, such as those described in U.S. Pat. No. 4,946,778, can also be adapted to produce single chain antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms, including other mammals, may be used to express humanized antibodies.

The above-described antibodies may be employed to isolate or to identify clones expressing the polypeptide or to purify the polypeptides by affinity chromatography. Antibodies against polypeptides of the present invention may also be employed to treat diseases of the invention, amongst others.

Polypeptides and polynucleotides of the present invention may also be used as vaccines. Accordingly, in a further aspect, the present invention relates to a method for inducing an immunological response in a mammal that comprises inoculating the mammal with a polypeptide of the present invention, adequate to produce antibody and/or T cell immune response, including, for example, cytokine-producing T cells or cytotoxic T cells, to protect said animal from disease, whether that disease is already established within the individual or not. An immunological response in a mammal may also be induced by a method comprises delivering a polypeptide of the present invention via a vector directing expression of the polynucleotide and coding for the polypeptide in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases of the invention. One way of administering the vector is by accelerating it into the desired cells as a coating on particles or otherwise. Such nucleic acid vector may comprise DNA, RNA, a modified nucleic acid, or a DNA/RNA hybrid. For use a vaccine, a polypeptide or a nucleic acid vector will be normally provided as a vaccine formulation (composition). The formulation may further comprise a suitable carrier. Since a polypeptide may be broken down in the stomach, it is preferably administered parenterally (for instance, subcutaneous, intramuscular, intravenous, or intradermal injection). Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions that may contain anti-oxidants, buffers, bacteriostats and solutes that render the formulation instonic with the blood of the recipient; and aqueous and non-aqueous sterile suspensions that may include suspending agents or thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use. The vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.

Polypeptides of the present invention have one or more biological functions that are of relevance in one or more disease states, in particular the diseases of the invention hereinbefore mentioned. It is therefore useful to to identify compounds that stimulate or inhibit the function or level of the polypeptide. Accordingly, in a further aspect, the present invention provides for a method of screening compounds to identify those that stimulate or inhibit the function or level of the polypeptide. Such methods identify agonists or antagonists that may be employed for therapeutic and prophylactic purposes for such diseases of the invention as hereinbefore mentioned. Compounds may be identified from a variety of sources, for example, cells, cell-free preparations, chemical libraries, collections of chemical compounds, and natural product mixtures. Such agonists or antagonists so-identified may be natural or modified substrates, ligands, receptors, enzymes, etc., as the case may be, of the polypeptide; a structural or functional mimetic thereof (see Coligan et al., Current Protocols in Immunology 1(2): Chapter 5 (1991)) or a small molecule.

The screening method may simply measure the binding of a candidate compound to the polypeptide, or to cells or membranes bearing the polypeptide, or a fusion protein thereof, by means of a label directly or indirectly associated with the candidate compound. Alternatively, the screening method may involve measuring or detecting (qualitatively or quantitatively) the competitive binding of a candidate compound to the polypeptide against a labeled competitor (e.g. agonist or antagonist). Further, these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide, using detection systems appropriate to the cells bearing the polypeptide. Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed. Further, the screening methods may simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide of the present invention, to form a mixture, measuring a ANIC-BP-1B activity in the mixture, and comparing the ANIC-BP-1B activity of the mixture to a control mixture which contains no candidate compound.

Polypeptides of the present invention may be employed in conventional low capacity screening methods and also in high-throughput screening (HTS) formats. Such HTS formats include not only the well-established use of 96- and, more recently, 384-well micotiter plates but also emerging methods such as the nanowell method described by Schullek et al, Anal Biochem., 246, 20–29, (1997).

Fusion proteins, such as those made from Fc portion and ANIC-BP-1B polypeptide, as hereinbefore described, can also be used for high-throughput screening assays to identify antagonists for the polypeptide of the present invention (see D. Bennett et al., J Mol Recognition, 8:52–58 (1995); and K. Johanson et al., J Biol Chem, 270(16):9459–9471 (1995)).

Screening Techniques

The polynucleotides, polypeptides and antibodies to the polypeptide of the present invention may also be used to configure screening methods for detecting the effect of added compounds on the production of mRNA and polypeptide in cells. For example, an ELISA assay may be constructed for measuring secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known in the art. This can be used to discover agents that may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues.

A polypeptide of the present invention may be used to identify membrane bound or soluble receptors, if any, through standard receptor binding techniques known in the art. These include, but are not limited to, ligand binding and crosslinking assays in which the polypeptide is labeled with a radioactive isotope (for instance, ¹²⁵I), chemically modified (for instance, biotinylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (cells, cell membranes, cell supernatants, tissue extracts, bodily fluids). Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy. These screening methods may also be used to identify agonists and antagonists of the polypeptide that compete with the binding of the polypeptide to its receptors, if any. Standard methods for conducting such assays are well understood in the art.

Examples of antagonists of polypeptides of the present invention include antibodies or, in some cases, oligonucleotides or proteins that are closely related to the ligands, substrates, receptors, enzymes, etc., as the case may be, of the polypeptide, e.g., a fragment of the ligands, substrates, receptors, enzymes, etc.; or a small molecule that bind to the polypeptide of the present invention but do not elicit a response, so that the activity of the polypeptide is prevented.

Screening methods may also involve the use of transgenic technology and ANIC-BP-1B gene. The art of constructing transgenic animals is well established. For example, the ANIC-BP-1B gene may be introduced through microinjection into the male pronucleus of fertilized oocytes, retroviral transfer into pre- or post-implantation embryos, or injection of genetically modified, such as by electroporation, embryonic stem cells into host blastocysts. Particularly useful transgenic animals are so-called “knock-in” animals in which an animal gene is replaced by the human equivalent within the genome of that animal. Knock-in transgenic animals are useful in the drug discovery process, for target validation, where the compound is specific for the human target. Other useful transgenic animals are so-called “knock-out” animals in which the expression of the animal ortholog of a polypeptide of the present invention and encoded by an endogenous DNA sequence in a cell is partially or completely annulled. The gene knock-out may be targeted to specific cells or tissues, may occur only in certain cells or tissues as a consequence of the limitations of the technology, or may occur in all, or substantially all, cells in the animal. Transgenic animal technology also offers a whole animal expression-cloning system in which introduced genes are expressed to give large amounts of polypeptides of the present invention.

Screening kits for use in the above described methods form a further aspect of the present invention. Such screening kits comprise:

-   -   (a) a polypeptide of the present invention;     -   (b) a recombinant cell expressing a polypeptide of the present         invention;     -   (c) a cell membrane expressing a polypeptide of the present         invention; or     -   (d) an antibody to a polypeptide of the present invention; which         polypeptide is preferably that of SEQ ID NO:2.

It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component.

Glossary

The following definitions are provided to facilitate understanding of certain terms used frequently hereinbefore.

“Antibodies” as used herein includes polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or another immunoglobulin expression library.

“Isolated” means altered “by the hand of man” from its natural state, i.e., if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living organism is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein. Moreover, a polynucleotide or polypeptide that is introduced into an organism by transformation, genetic manipulation or by any other recombinant method is “isolated” even if it is still present in said organism, which organism may be living or non-living.

“Polynucleotide” generally refers to any polyribonucleotide (RNA) or polydeoxribonucleotide (DNA), which may be unmodified or modified RNA or DNA. “Polynucleotides” include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, “polynucleotide” refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term “polynucleotide” also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.

“Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications may be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. “Polynucleotide” also embraces relatively short polynucleotides, often referred to as oligonucleotides.

“Polypeptide” refers to any polypeptide comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. “Polypeptide” refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. “Polypeptides” include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications may occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present to the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from post-translation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, biotinylation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination (see, for instance, Proteins—Structure and Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York, 1993; Wold, F., Post-translational Protein Modifications: Perspectives and Prospects, 1–12, in Post-translational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York, 1983; Seifter et al., “Analysis for protein modifications and nonprotein cofactors”, Meth Enzymol, 182, 626–646, 1990, and Rattan et al., “Protein Synthesis: Post-translational Modifications and Aging”, Ann NY Acad Sci, 663, 48–62, 1992).

“Fragment” of a polypeptide sequence refers to a polypeptide sequence that is shorter than the reference sequence but that retains essentially the same biological function or activity as the reference polypeptide. “Fragment” of a polynucleotide sequence refers to a polynucloetide sequence that is shorter than the reference sequence of SEQ ID NO:1.

“Variant” refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains the essential properties thereof. A typical variant of a polynucleotide differs in nucleotide sequence from the reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in amino acid sequence from the reference polypeptide. Generally, alterations are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, insertions, deletions in any combination. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. Typical conservative substitutions include Gly, Ala; Val, Lle, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe and Tyr. A variant of a polynucleotide or polypeptide may be naturally occurring such as an allele, or it may be a variant that is not known to occur naturally. Non-naturally occurring *variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis. Also included as variants are polypeptides having one or more post-translational modifications, for instance glycosylation, phosphorylation, methylation, ADP ribosylation and the like. Embodiments include methylation of the N-terminal amino acid, phosphorylations of serines and threonines and modification of C-terminal glycines.

“Allele” refers to one of two or more alternative forms of a gene occuring at a given locus in the genome.

“Polymorphism” refers to a variation in nucleotide sequence (and encoded polypeptide sequence, if relevant) at a given position in the genome within a population.

“Single Nucleotide Polymorphism” (SNP) refers to the occurence of nucleotide variability at a single nucleotide position in the genome, within a population. An SNP may occur within a gene or within intergenic regions of the genome. SNPs can be assayed using Allele Specific Amplification (ASA). For the process at least 3 primers are required. A common primer is used in reverse complement to the polymorphism being assayed. This common primer can be between 50 and 1500 bps from the polymorphic base. The other two (or more) primers are identical to each other except that the final 3′ base wobbles to match one of the two (or more) alleles that make up the polymorphism. Two (or more) PCR reactions are then conducted on sample DNA, each using the common primer and one of the Allele Specific Primers.

“Splice Variant” as used herein refers to cDNA molecules produced from RNA molecules initially transcribed from the same genomic DNA sequence but which have undergone alternative RNA splicing. Alternative RNA splicing occurs when a primary RNA transcript undergoes splicing, generally for the removal of introns, which results in the production of more than one mRNA molecule each of that may encode different amino acid sequences. The term splice variant also refers to the proteins encoded by the above cDNA molecules.

“Identity” reflects a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, determined by comparing the sequences. In general, identity refers to an exact nucleotide to nucleotide or amino acid to amino acid correspondence of the two polynucleotide or two polypeptide sequences, respectively, over the length of the sequences being compared.

“% Identity”—For sequences where there is not an exact correspondence, a “% identity” may be determined. In general, the two sequences to be compared are aligned to give a maximum correlation between the sequences. This may include inserting “gaps” in either one or both sequences, to enhance the degree of alignment. A % identity may be determined over the whole length of each of the sequences being compared (so-called global alignment), that is particularly suitable for sequences of the same or very similar length, or over shorter, defined lengths (so-called local alignment), that is more suitable for sequences of unequal length.

“Similarity” is a further, more sophisticated measure of the relationship between two polypeptide sequences. In general, “similarity” means a comparison between the amino acids of two polypeptide chains, on a residue by residue basis, taking into account not only exact correspondences between a between pairs of residues, one from each of the sequences being compared (as for identity) but also, where there is not an exact correspondence, whether, on an evolutionary basis, one residue is a likely substitute for the other. This likelihood has an associated “score” from which the “% similarity” of the two sequences can then be determined.

Methods for comparing the identity and similarity of two or more sequences are well known in the art. Thus for instance, programs available in the Wisconsin Sequence Analysis Package, version 9.1 (Devereux J et al, Nucleic Acids Res, 12, 387–395, 1984, available from Genetics Computer Group, Madison, Wis., USA), for example the programs BESTFIT and GAP, may be used to determine the % identity between two polynucleotides and the % identity and the % similarity between two polypeptide sequences. BESTFIT uses the “local homology” algorithm of Smith and Waterman (J Mol Biol, 147,195–197, 1981, Advances in Applied Mathematics, 2, 482–489, 1981) and finds the best single region of similarity between two sequences. BESTFIT is more suited to comparing two polynucleotide or two polypeptide sequences that are dissimilar in length, the program assuming that the shorter sequence represents a portion of the longer. In comparison, GAP aligns two sequences, finding a “maximum similarity”, according to the algorithm of Neddleman and Wunsch (J Mol Biol, 48, 443–453, 1970). GAP is more suited to comparing sequences that are approximately the same length and an alignment is expected over the entire length. Preferably, the parameters “Gap Weight” and “Length Weight” used in each program are 50 and 3, for polynucleotide sequences and 12 and 4 for polypeptide sequences, respectively. Preferably, % identities and similarities are determined when the two sequences being compared are optimally aligned.

Other programs for determining identity and/or similarity between sequences are also known in the art, for instance the BLAST family of programs (Altschul S F et al, J Mol Biol, 215, 403410, 1990, Altschul S F et al, Nucleic Acids Res., 25:389–3402, 1997, available from the National Center for Biotechnology Information (NCBI), Bethesda, Md., USA and accessible through the home page of the NCBI at www.ncbi.nim.nih.gov) and FASTA (Pearson W R, Methods in Enzymology, 183, 63–99, 1990; Pearson W R and Lipman D J, Proc Nat Acad Sci USA, 85, 2444–2448,1988, available as part of the Wisconsin Sequence Analysis Package).

Preferably, the BLOSUM62 amino acid substitution matrix (Henikoff S and Henikoff J G, Proc. Nat. Acad. Sci. USA, 89, 10915–10919, 1992) is used in polypeptide sequence comparisons including where nucleotide sequences are first translated into amino acid sequences before comparison.

Preferably, the program BESTFIT is used to determine the % identity of a query polynucleotide or a polypeptide sequence with respect to a reference polynucleotide or a polypeptide sequence, the query and the reference sequence being optimally aligned and the parameters of the program set at the default value, as hereinbefore described.

“Identity Index” is a measure of sequence relatedness which may be used to compare a candidate sequence (polynucleotide or polypeptide) and a reference sequence. Thus, for instance, a candidate polynucleotide sequence having, for example, an Identity Index of 0.95 compared to a reference polynucleotide sequence is identical to the reference sequence except that the candidate polynucleotide sequence may include on average up to five differences per each 100 nucleotides of the reference sequence. Such differences are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion. These differences may occur at the 5′ or 3′ terminal positions of the reference polynucleotide sequence or anywhere between these terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. In other words, to obtain a polynucleotide sequence having an Identity Index of 0.95 compared to a reference polynucleotide sequence, an average of up to 5 in every 100 of the nucleotides of the in the reference sequence may be deleted, substituted or inserted, or any combination thereof, as hereinbefore described. The same applies mutatis mutandis for other values of the Identity Index, for instance 0.96, 0.97, 0.98 and 0.99.

Similarly, for a polypeptide, a candidate polypeptide sequence having, for example, an Identity Index of 0.95 compared to a reference polypeptide sequence is identical to the reference sequence except that the polypeptide sequence may include an average of up to five differences per each 100 amino acids of the reference sequence. Such differences are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion. These differences may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between these terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence. In other words, to obtain a polypeptide sequence having an Identity Index of 0.95 compared to a reference polypeptide sequence, an average of up to 5 in every 100 of the amino acids in the reference sequence may be deleted, substituted or inserted, or any combination thereof, as hereinbefore described. The same applies mutatis mutandis for other values of the Identity Index, for instance 0.96, 0.97, 0.98 and 0.99.

The relationship between the number of nucleotide or amino acid differences and the Identity Index may be expressed in the following equation: n _(a) ≦X _(a)−(X _(a) ·I), in which:

-   -   n_(a) is the number of nucleotide or amino acid differences,     -   X_(a) is the total number of nucleotides or amino acids in SEQ         ID NO:1 or SEQ ID NO:2, respectively,     -   I is the Identity Index,     -   ·is the symbol for the multiplication operator, and     -   in which any non-integer product of X_(a) and I is rounded down         to the nearest integer prior to subtracting it from x_(a).

“Homolog” is a generic term used in the art to indicate a polynucleotide or polypeptide sequence possessing a high degree of sequence relatedness to a reference sequence. Such relatedness may be quantified by determining the degree of identity and/or similarity between the two sequences as hereinbefore defined. Falling within this generic term are the terms “ortholog”, and “paralog”. “Ortholog” refers to a polynucleotide or polypeptide that is the functional equivalent of the polynucleotide or polypeptide in another species. “Paralog” refers to a polynucleotideor polypeptide that within the same species which is functionally similar.

“Fusion protein” refers to a protein encoded by two, unrelated, fused genes or fragments thereof. Examples have been disclosed in U.S. Pat. Nos. 5,541,087, 5,726,044, EP 574395, EP 493418 and EP 0232 262. In the case of Fc-ANIC-BP-1B, employing an immunoglobulin Fc region as a part of a fusion protein is advantageous for performing the functional expression of Fc-ANIC-BP-1B, to improve pharmacokinetic properties of such a fusion protein when used for therapy and to generate a dimeric Fc-ANIC-BP-1B. The Fc-ANIC-BP-1B DNA construct comprises in 5′ to 3′ direction, a secretion cassette, i.e. a signal sequence that triggers export from a mammalian cell, DNA encoding an immunoglobulin Fc region fragment, as a fusion partner, and a DNA encoding Fc-ANIC-BP-1B. In some uses it would be desirable to be able to alter the intrinsic functional properties (complement binding, Fc-Receptor binding) by mutating the functional Fc sides while leaving the rest of the fusion protein untouched or delete the Fc part completely after expression.

All publications and references, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference in their entirety as if each individual publication or reference were specifically and individually indicated to be incorporated by reference herein as being fully set forth. Any patent application to which this application claims priority is also incorporated by reference herein in its entirety in the manner described above for publications and references.

Figure Legends:

FIG. 1

Representative mRNA differential display gel with differentially regulated band in the cerebellar hemisphere contralateral to the side of injury. The arrow marks the differentially expressed band.

FIG. 2

RT-PCR with the 270 bp gene fragment of rat Mo25 in the cerebellum after TBI at cycles 5,10,15,20,25 and 30, respectively. Notice the strong expression in the L2 lane at the cycles 20, 25 and 30.

L2: cDNA of TBI rat non lesioned side; R2: cDNA of TBI rat lesioned side.

FIG. 3

Dot blot analysis with radioactive ³²P marked 270 bp gene fragment of rat Mo25 with cerebellum mRNA of rat after TBI. Six individual rats were examined, 3 TBI treated and 3 sham operated animals.

FIG. 4

Multiple tissue Northern blots (Clontech Laboratories Inc, Palo Alto, Calif., USA), with radioactive ³²P marked 270 bp gene fragment of rat Mo25 hybridised on 8 rat tissues

FIG. 5

In situ hybridisation of rat brain sections. Sence and antisence probes specific for SICCBP and Mo25 have been used. Strong signals lighting up myelinated nerve tructs (Corpus Callosum. Tractus opticus, tractus olfactorius intermedius, cerebellum commissura anterior) have been monitored using the antisence probes.

FURTHER EXAMPLES Example 1

Traumatic Brain Injury Model

The identification of genes up or down regulated in response to traumatic brain injury (TBI), was studied in rats rats using the lateral fluid method. A moderate TBI centered on the right parietal cortex was induced with the lateral fluid percussion method in male Sprague-Dawley rats. Five days after TBI, rats were sacrificed and dissected brain tissue was analyzed by differential display. Upregulation of protein in cerebellum of traumatized brains was confirmed by RT-PCR.

Control rats were anesthetized and the temporal muscle was retracted, but no craniotomy was performed. After a five day survival, all rats were anesthetized, killed, and the brain was dissected and frozen in liquid nitrogen.

A second series of TBI rats were used for histochemical and immunohistochemical staining. One week after TBI, these rats were anesthetized and perfused with saline followed by 3% paraformaldehyde. The brains were cut on a freezing microtome into 30 μm coronal sections.

Example 2

Immunohistochemistry

Cerebellar sections were labeled with monoclonal antibodies against the calcium binding protein. The immuno complex was visualized using the avidin-biotin/DAB method. As a positive control the Calbindin-D (28 kD) has been used as a reliable marker of cerebellar Purkinje cells.

Example 3

In Situ Hybridisation

Oligodeoxynucleotides selected from Sequence No.1 (sence, antisence) have been designed according to standard methods apperent to those skilled in the art. These probes have been used for in situ hybridisation in rat brain tissue sections according to methods known in the art. Autoradiograph have been visualized after 5 day exposure on Kodak BioMax-Film.

Example 4

RNA-Isolation from TBI Rats

mRNA differential display has been developed as a method to identify and analyze altered gene expression at the mRNA level in any eukaryotic cell (Liang and Pardee, Science 257, 967,1992). In this invention we used this method to study genes up or down regulated in response to traumatic brain injury, in order to obtain a better insight into the molecular effects of CNS injury (den Daas et al.; Meeting of the American Neuroscience Society Washington D.C., USA; 1998). The animal model for traumatic brain injury is called the lateral fluid percussion model and is performed as follows: a moderate traumatic brain injury centered on the right parietal cortex of the brain was induced with the lateral fluid percussion method in male Sprague-Dawley rats. Control rats were anesthetized and the temporal muscle was retracted, but no craniotomy was performed. After a five-day survival, rats were anesthetized, killed, the brain was dissected and frozen in liquid nitrogen. Whole brains were homogenized and total RNA was isolated (Sambrook et al. 1989).

Differential Display

Obtained RNA was analyzed by RNA differential display. Reverse transcription of mRNAs was performed using oligo-dT primers with two additional nucleotides in all possible combinations (downstream primers; 13 mere), thus anchoring the reaction to the beginning of the poly (A) tail.

Amplification of the cDNA was conducted with the same 3′primer and a second decamer arbitrary 5′primer (upstream primer). Amplification products were analyzed on a nondenaturing 10% polyacrylamide gel (Amersham Pharmacia Biotech, Germany). DNA was visualized by silver staining. After staining, gels were dried for one hour at room temperature (FIG. 1). Differentially regulated bands were cut out from the gel. DNA was eluted, reamplified and subcloned into pCR2.1 vector (Invitrogen, USA). Subcloned fragments were sequenced by the Sanger method (Sanger F., et al, PNAS, USA 74, 5463–5467) and sequences were compared to genomic databases. Validation of the obtained gene fragment was done with reverse transcription PCR (RT-PCR). For confirmation of the differentially expressed rat Mo25, sequence was analyzed by RT-PCR using the Titan one tube RT-PCR system (Boehringer Mannheim, Germany) with specific 19 mere and 21 mere primers. One μg of total RNA from control and TBI animals were used for the RT-PCR. More gene validation has been performed using the dotblot technique with probes from rat Mo25 and Northern blot analysis performed with probes from human Mo25.

Reverse Transcription PCR (RT-PCR)

For confirmation of the differentially expressed stroke induced calcium binding protein, sequence was analyzed by RT-PCR using the Titan One Tube RT-PCR system (Boehringer Mannheim, Germany) with specific 19 mere and 21 mere primers. One μg of total RNA from control and TBI animals were used for the RT-PCR.

Real Time PCR

Distribution of ANIC-BP-1B in the rat brain after head trauma was examined by the Real Time TaqMan PCR technique in a ABI prism 7700 sequence detection system (PE, Applied Biosystems, Germany). With this technique, absolute concentrations of mRNA can be measured with high sensitivity. Special primers with a length of 25 and 29 bp and a 32 mere TaqMan probe (reporter dye: FAM/quencher dye: TAMRA) were designed.

Ca2+ binding

Protein separated by SDS-PAGE was blotted onto nitro cellulose membrane and assayed for binding of radiolabled Ca 2+ using the method described by Maruyama, K. et al. (J. Biochem. 95:511–519, 1984). After incubation the surplus of isotope was washed off and the membrane was exposed to Kodak XOMAT.TM film for an appropriate time. A band developed at the position of the binding protein. The presence of the binding protein was confirmed by using antibody specific for the binding protein and applying that antibody by the western blot method that is well known in the art. 

1. An isolated human polypeptide selected from the group consisting of: (a) an isolated polypeptide encoded by a polynucleotide comprising of SEQ ID NO: 1; (b) an isolated polypeptide comprising a polypeptide sequence comprising at least 95% identity along its entire length to the polypeptide sequence of SEQ ID NO:2 and which is encoded by a polynucleotide which hybridizes to the complement of SEQ ID NO:1 under stringent hybridization conditions comprising overnight incubation at 42° C. in a solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA; followed by washing the filters in 0.1× SSC at 65° C., wherein said polypeptide has calcium binding activity; c) an isolated polypeptide comprising at least 95% identity along its entire length to the polypeptide sequence of SEQ ID NO:2 and which is encoded by a polynucleotide which hybridizes to the complement of SEQ ID NO:1 under stringent hybridization conditions comprising overnight incubation at 42° C. in a solution comprising: 50% formamide, 5× SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA; followed by washing the filters in 0.1× SSC at 65° C., wherein said polypeptide has calcium binding activity; and d) the polypeptide sequence of SEQ ID NO:2.
 2. The isolated polypeptide as claimed in claim 1 comprising the polypeptide sequence of SEQ ID NO:2.
 3. The isolated polypeptide as claimed in claim 1 which consists of the polypeptide sequence of SEQ ID NO:2.
 4. A fusion protein comprising an Immunoglobulin Fc-region and a polypeptide of claim
 1. 5. The isolated polypeptide as claimed in claim 1, which is encoded by a polynucleotide comprising the sequence of SEQ ID NO:
 1. 6. The isolated polypeptide as claimed in claim 1, which is an isolated polypeptide comprising a polypeptide sequence comprising at least 95% identity along its entire length to the polypeptide sequence of SEQ ID NO: 2 and which is encoded by a Polynucleotide which hybridizes to the complement of SEQ ID NO:1 under stringent hybridization conditions comprising overnight incubation at 42° C. in a solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA; followed by washing the filters in 0.1×SSC at 65° C., wherein said polypeptide has calcium binding activity.
 7. The isolated polypeptide as claimed in claim 1, which is an isolated polypeptide comprising at least 95% identity along its entire length to the polypeptide sequence of SEQ ID NO: 2 and which is encoded by a polynucleotide which hybridizes to the complement of SEQ ID NO: 1 under stringent hybridization conditions comprising overnight incubation at 42° C. in a solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA; followed by washing the filters in 0.1×SSC at 65° C., wherein said polypeptide has calcium binding activity. 